Method for preparing two-layer bicomposite collagen material for preventing post-operative adhesions

ABSTRACT

A bicomposite material based on collagen is prepared which has two closely bound layers and is biocompatible, non-toxic, hemostatic and biodegradable in less than a month, and can be used in surgery to achieve hemostasis and prevent post-surgical adhesion. To prepare the material, a solution of collagen or gelatin, which may contain glycerine and a hydrophilic additive such as polyethylene glycol or a polysaccharide, is poured onto an inert support to form a layer 30 μm to less than 100 μm thick. Then a polymeric porous fibrous layer is applied during gelling of the collagen or gelatin, and the resultant material is dried. The polymeric porous fibrous layer may be made of collagen or a polysaccharide, and have a density of not more than 75 mg/cm 2 , a pore size from 30 μm to 300 μm and a thickness of 0.2 cm to 1.5 cm.

FIELD OF THE INVENTION

The present invention concerns a bicomposite material based on collagen,which is biocompatible, non-toxic and biodegradable, comprising uniquelyor mainly a layer forming a collagenic film and a layer forming afibrous polymer compress or sponge with a high level of porosity.

The material according to the invention can be used in surgery, notablyin visceral surgery, and is specifically applied for the simultaneousachievement of hemostasis and prevention of post-surgical adhesion,while promoting the healing of the injured tissue.

DESCRIPTION OF THE RELATED ART

Patents FR-A-2 628 634 and U.S. Pat. No. A-5,201,745 (IMEDEX) describepatches for use in visceral surgery made of a biomaterial consisting oftwo layers of collagen superposed and closely associated, these being aporous adhesive layer of fibrous collagen and a collagen film orcollagenic material such as gelatine.

In this type of material, the film seals the membrane or patch andincreases mechanical cohesion, also helping to prevent the formation ofpost-operative adhesions. The porous layer of fibrous collagen notablyplays the part of a hemostatic compress.

A double-layered collagenic membrane has been proposed in patentapplications EP-A-O 686 402 and WO 96/08277 (COLETICA) with the aim ofobtaining anti-adhesive properties.

The collagens and collagenic materials used in such patches or membranesmay be obtained from native collagen or from different types ofatelocollagens or pepsin-treated collagens, notably type I bovinecollagens, and type I, III, III+I and IV human collagens. Thesecollagens can be partly oxidized, for example to increase their adhesivepower, and the layer forming the film may include other materials, mixedwith the collagenic material, used, for example to strengthen itsmechanical resistance and improve its anti-adhesion properties. It isnot easy to produce these patches or membranes, however. Indeed, on theone hand it is essential to guarantee an excellent bond between thelayer forming the film and the layer forming the fibrous compress, whileretaining each layer's individuality on the other. Also, when the layerof fibrous material is brought into contact with the liquid collagenicmaterial destined to form the film, on contact with the liquid, thecollagenic fibres tend to become impregnated so that an excellent bondis indeed obtained between the two layers but it is very difficult tocontrol formation of the film and respect the porosity of the supportinglayer.

For this purpose, it has been proposed (FR-A-2 628 634), to pour thecollagenic material which is to form the film, onto a layer of fibrouscollagen which has first been slightly compressed to limitinterpenetration between the two layers.

It has also already been proposed (EP-A-O 686 402) to freeze the porousfibrous layer so that it is hydrated and impermeable and pour the liquidcollagenic material destined to form the film onto this layer so as toeliminate interpenetration between the two layers, but this level ofprevention of interpenetration gives rise to cohesion defects. Theprocess described also gives rise to a two-layer collagengelatinemembrane which has been dried or freeze-dried in one piece, whichprevents an impermeable film and a highly porous layer from being formedsimultaneously. It is also recommended to compress this membrane.

Hemostatic sponges composed of native bovine collagen are commerciallyavailable, as for example Colgen® (Immuno AG), Pangen® (Fournier) andSurgicoll® (Biodynamics); but these are not covered on one side with animpermeable film, acting as a barrier and they have severaldisadvantages:

-   -   i) left in the body, they can generate adhesions;    -   ii) the blood diffuses through preferential routes in the        compress, reducing the area of contact of the collagen with the        platelets and consequently the hemostatic effect of the        compress;    -   iii) they no longer have a hemostatic effect on strongly        bleeding wounds (ruptures arterioles for example), because the        blood passes through the compress;    -   iv) generally produced from acid collagen, they are difficult to        handle because they strongly stick to surgical instruments or        latex gloves.

Other more complex products, such as TachoComb® (Nycomed) combiningcollagen, fibrinogen, thrombin and aprotinin provide better hemostasisthan collagen sponges, but these products are likely to facilitate thedevelopment of post-operative adhesions. They contain thermolabileenzymes and must be stored between 2 and 8° C. The multiplication ofcomponents of human or animal origin is also a handicap, because ofproblems of traceability and registration linked to these products,leading to prohibitive excess cost.

From the point of view of preventing post-operative adhesions, this isparticularly difficult with haemorrhagic wounds, especially wherebleeding is widespread (Buckman et al., J. Surg. Res., 1976, 20 1-5;Wiseman et al., J. Reprod. Med., 1992, 37, 766-770). Bleeding fromwounds strongly affects the efficacy of the products marketed and usedto prevent adhesion, such as INTERCEED® TC7 (Johnson & Johnson) (Wisemanet al., J. Reprod. Med., 1992, 37, 766-770). Indeed it can lead to thedeposit of fibrin on the anti-adhesive film and then facilitate thedevelopment of post-operative adhesions. This results in the necessityto perform the most complete hemostasis possible, using thrombin or anyother technique, before applying products such as INTERCEED® TC7 tohaemorrhagic wounds. Therefore to prevent adhesion it is advantageous todevelop materials which also have hemostatic properties.

SUMMARY OF THE INVENTION

The present invention therefore aims to considerably perfect thepreviously described bicomposite collagenic materials, and to improvetheir hemostatic properties considerably, while retaining and, ifnecessary, even improving their properties which aim to preventpost-operative adhesions.

The invention also aims to provide a hemostatic bicomposite collagenicmaterial which can, in addition, prevent post-operative adhesions andfacilitate healing.

Another of the invention's aims is to produce such a material whichparticularly promotes colonization by the body's specific cells and islikely to be completely biodegradable within a short time and easy tocontrol by making simple changes to the manufacturing process.

The invention also aims to provide a biocompatible bicomposite materialwhich is non-toxic and not sticky to the touch when dry, to facilitatehandling, but which can develop adhesive properties in a physiologicalenvironment, in particular in contact with blood.

Another of the invention's aim is to provide a particularly economicprocess to obtain such a bicomposite material.

Therefore the invention aims to produce a bicomposite collagenicmaterial which is biocompatible, non-toxic and biodegradable in lessthan a month, characterized in that it comprises solely or principallytwo closely linked layers, these being a layer forming a film based on acollagenic constituent, notably collagen which has at least partiallylost its helical structure, or gelatine, and a layer forming a porouscompress, substantially uncompacted, based on a polymer constituent.

As well as the collagenic constituent, the film preferably comprises atleast one macromolecular hydrophilic additive which does not reactchemically with collagen.

The second layer can be made of a porous compress, substantiallyuncompacted, of non-denatured collagen.

The invention also aims to provide a preferred process for producingthese materials.

This process is based on the discovery that, when a liquid solutionbased on a collagenic constituent destined to form a film is left togel, there is an instant, during gelling, when the porous layer ofpolymer constituent forming the compress can be laid on the surface ofthe gelling material, and the under part of the said porous layer partlypenetrates the gel, while at least partly retaining a structure whichguarantees almost perfect adhesion between the film to be constitutedand the porous layer, while preserving almost all the individualproperties of the porous layer and the film.

The inventors noted most surprisingly that:

-   -   the collagen film can be formed by dehydration of the liquid        layer of collagen in spite of the presence of a freeze-dried        porous layer on top of it;    -   the upper porous layer is not degraded or changed by association        with the film in the process of formation.

The invention therefore aims to provide a process for obtaining abicomposite material according to the invention, characterized in that asolution of collagenic constituent is poured onto a suitable inertsupport, to a thickness destined to form a film, and in that asubstantially uncompacted compress made of a polymer constituent isapplied onto the said solution during gelification, and then in that thematerial obtained is dried or left to dry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph illustrating structures of the bicompositematerial depicting a specimen of Example 7 in accordance with thepresent invention; and

FIG. 2 is a photograph showing a specimen of Example 7 made from thecompress as in Example 3 with the film being produced as in Example 8 inaccordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The process according to the invention will be described in greaterdetail below:

To implement this process, an aqueous solution of the collagenicconstituent destined to form the film of the above-mentioned bicompositematerial is prepared.

According to the invention, the term “collagenic constituent” preferablydesignates collagen which has at least partially lost its helicalstructure through heating or any other method, or gelatine.

The term “gelatine” here includes commercial gelatine made of collagenwhich has been denatured by heating and in which the chains are at leastpartially hydrolyzed (molecular weight lower than 100 kDa).

The collagenic constituent used for the purposes of the invention ispreferably formed of non-hydrolyzed collagen, mainly composed of achains (molecular weight around 100 kDa).

In the context of the invention α chains means complete a chains orfragments of these complete α chains produced by the loss of a smallnumber of amino acids.

The term “non-hydrolyzed” as used according to the invention means thatless than 10% of the collagenic chains has a molecular weight belowabout 100 kDa.

If heating is used to denature the helical structure of the collagen,the heating must be moderate and provided under gentle conditions so asto avoid degradation by hydrolytic cleavage of the gelatine thus formed.

Commercial gelatine can be used for the invention but is not preferred.

The collagen used can be of human or animal origin. It may particularlybe type I bovine collagen, or type I or type III human collagen ormixtures in any proportions of the last two types.

Native collagen is used by preference, in acid solution or afterprocessing, to eliminate the telopeptides, notably by pepsin digestion.

The collagen can also be modified by oxidative cleavage. For thispurpose periodic acid or one of its salts can be used, applying thetechnique described by M. TARDY et al. (FR-A-2 601 371 and U.S. Pat. No.4,931,546).

It is recalled briefly that this technique consists of mixing thecollagen in acid solution with a solution of periodic acid or one of itssalts at a concentration of between 1 and 10⁻⁵ M, preferably between 510⁻³ and 10⁻¹ M, at a temperature of between 10 and 25° C. for 10minutes to 72 hours.

This process breaks down some of the collagen's components, these beinghydroxylysine and the sugars, thus creating reactive sites withoutcausing crosslinking.

The oxidative cleavage of collagen allows moderate cross-linking laterin the collagenic material but the invention does not exclude thepossibility of providing this function by other means of moderatecross-linking, for example by beta or gamma irradiation, or other agentsof moderate cross-linking, for example chemical reagents at suitably lowand non-toxic doses.

For some applications, the film part of the bicomposite materialaccording to the invention, is made of collagen which is not oxidized ora mixture in any proportions of non-oxidized and oxidized collagens.

In a preferred embodiment of the invention, a solution of collagenicconstituent as defined above is used, and this may be partially orcompletely modified by oxidative cleavage, giving a collagenconcentration of 5 to 50 g/l. The collagen or gelatine concentration ispreferably 30 g/l.

The solution of oxidized collagen, non-oxidized collagen or a mixturethereof, thus prepared, is heated, for example to a temperature inexcess of 37° C., preferably to a temperature of between 40 and 50° C.,for at least one hour. This results in the collagen's helical structurebeing at least partially denatured.

A final preparation can notably be obtained which is similar to gelatinebut with a molecular weight of elementary chains equal or greater than100 kDa.

Heating the collagen solution to a temperature above 37° C. leads to thegradual loss of the collagen's helical structure, but the invention doesnot exclude the possibility of achieving this by other physical orchemical means, for example by ultrasonication, or by the addition ofchaotropic agents.

According to a variant of the invention, at least one macromolecularhydrophilic additive is added to the previous preparation, this beingpreferably chemically unreactive with the collagenic constituent.

“Chemically unreactive with the collagenic constituent” here means ahydrophilic compound which is not likely to react with the collagenpresent, notably which does not form covalent bonds with it duringcross-linking.

The macromolecular hydrophilic additive according to the inventionadvantageously has a molecular weight in excess of 3,000 Daltons.

It may consist of synthetic hydrophilic polymers, preferably of amolecular weight between 3,000 and 20,000 Daltons. Polyethylene glycolis particularly preferred.

It may also consist of polysaccharides, of which starch, dextran andcellulose can be mentioned.

Oxidized forms of these polysaccharides can also be used, revealingcarboxylic functions in these molecules.

Mucopolysaccharides can also be used for the purposes of the invention,but are not preferred because their particular animal origin makes themdifficult to prepare so that they meet the standards of traceability.

The hydrophilic additive is selected according to various parameters,notably concerning its application, price, safety, biodegradabilityand/or ease of elimination.

The concentration of hydrophilic additive(s) is 2 to 10 times less thanthat of the collagenic constituent.

According to a variant execution of the invention, glycerine is added tothe mixture of collagenic constituent/hydrophilic additive(s).

In this case, the concentration of glycerin is advantageously between 3and 8 g/l, not exceeding one third of the collagenic constituentconcentration.

In the collagenic preparation, the concentration of collagenicconstituent, hydrophilic additive(s) and glycerine, when present, arepreferably between 2 and 10% for the collagenic constituent, 0.6 and 4%for the hydrophilic additive(s) and 0.3 and 2.5% for glycerinerespectively.

The collagenic preparation is fluidised at a temperature of 30 to 50° C.

It is advantageously neutralized to a neutral pH to avoid hydrolyzingthe collagen by heating and to obtain a film of physiological pH whilepermitting pre-cross-linking of the collagen if the mixture containsoxidized collagen as indicated previously.

For implementation of the process according to the invention, asubstantially non compacted porous compress, based on a polymerconstituent is also prepared.

The term “polymer constituent” according to the invention means afibrous, non toxic polymer with hemostatic and/or heating properties. Itmay be non-denatured collagen or collagen which has at least partiallylost its helical structure through heating or any other method,consisting mainly of non-hydrolyzed α chains, of molecular weight closeto 100 kDa. It may also consist of polysaccharides such as chitin orchitosan, or polysaccharides modified by oxidation of alcohol functionsinto carboxylic functions such as oxidized cellulose.

The term “non-denatured collagen” means collagen which has not lost itshelical structure.

The collagen used for this second layer of bicomposite materialaccording to the invention, consists of native collagen oratelocollagen, notably as obtained through pepsin digestion and/or aftermoderate heating as defined previously.

These may have been previously chemically modified by oxidation,methylation, succinylation or any other known process.

The origin and type of collagen are as indicated for the film describedabove.

The term “substantially non compacted porous compress” means a compressmade of polymer fibres with a porous structure such as is obtained byfreeze-drying for example, or an even more porous compress which canthen have been slightly compacted.

Defined in another form, the said layer forming a porous compress has adensity of not more than 75 mg/cm² and preferably below 20 mg/cm².

The porosity of these materials is illustrated in FIGS. 1 and 2.

The size of the pores varies from 20 to 300 μm and is generally between100 and 200 μm.

The porous compress can be obtained preferably by freeze-drying, from anaqueous acid solution of collagen at a concentration of 2 to 50 g/l anda temperature of 4 to 25° C. The concentration of collagen is preferably10 g/l.

This solution is advantageously neutralized to a pH of around 7 to 8.

The porous compress can also be obtained by freeze-drying a fluid foamprepared from a solution of collagen or heated collagen, emulsified inthe presence of a volume of air in variable respective quantities(volume of air:water varying from 1 to 10).

The porous fibrous layer made of a polymer constituent is preferably atleast 0.2 cm thick and is particularly preferred between 0.3 and 1.5 cmthick.

The actual bicomposite material is prepared by assembling thefilm-forming layer and the porous compress as detailed below.

In its simplest method of implementation, the process according to theinvention involves pouring the solution of collagenic constituent,destined to form the film, possibly containing the hydrophilicadditive(s) and glycerine, onto an adequate, substantially flat support,distributing it evenly.

The support is inert in that it does not react with the above-mentionedcomponents and is not involved in the cross-linking process. It ispreferably hydrophobic, for example, PVC or polystyrene.

However, this support can also consist of a strippable material whichwill remain slightly adhesive and which can then be separated at thetime of surgical use.

This support may itself also consist of a film, for example driedcollagen, onto which the solution is poured, or a layer of collagenicmaterial gel in a distinctly more advanced state of gelification.

The density of the thin layer applied is preferably between 0.1 and 0.3g/cm².

This collagenic solution is poured at a temperature advantageouslybetween 4 and 30° C., and preferably between 18 and 25° C.

This solution is left to gel and a porous compress prepared as indicatedabove is applied to the said solution during gelification.

Application of the porous layer onto the solution during gelificationmeans laying the porous layer onto the gel, with application continuingby simple gravity or optionally, by slight compression but not enough tocause any significant compaction of the porous layer.

The moment at which the porous layer is applied to the solution duringgelification is such that the gel is still soft and allows the porouslayer or compress to penetrate over a distance which is advantageouslyaround 0.05 to 2 mm and preferably around 0.1 to 0.5 mm.

This moment can be determined empirically by applying compresses or bitsof compresses to the gel at various times.

Generally, when the solution which is gelling is at a temperature ofbetween 4 and 30° C., the porous layer is applied 5 to 30 minutes afterthe solution has been poured over the surface holding it.

It is left to dry or dried in order to obtain the bicomposite materialaccording to the invention.

When the collagenic solution destined to form the film includes oxidizedcollagen, it is polymerized while the bicomposite material is drying.

This drying occurs favourably at a temperature of 4 to 30° C.,preferably between 18 and 25° C.

The material can be dried in a jet of sterile air if necessary.

After drying, the bicomposite material according to the invention can beseparated from its support. In a variant, it may include or incorporatea film or layer of collagenic material onto which the collagenicsolution has been poured.

The process described above may be implemented in a similar way usingother types of hemostatic compresses, notably compresses such as areavailable commercially. Examples of these are compresses based onoxidized cellulose (Surgicel® or Interceed® compresses) or those basedon chitin or chitosan.

The bicomposite material according to the invention is stable at ambienttemperature and remains stable for long enough to be handled attemperatures which may rise to 37-40° C.

The film of collagenic material is preferably less than 100 μm thick,and more preferably between 30 and 75 μm.

The porous compress is preferably between 0.2 cm and 1.5 cm thick, andstill more preferably between 0.3 cm and 1.2 cm.

According to the envisaged applications, the bicomposite materialconforming to the invention can be subjected to various routineprocesses such as sterilization, etc.

Sterilization is favourably provided by irradiation with beta(electronic irradiation) or gamma (irradiation using radioactive cobalt)rays.

The bicomposite material according to the invention can be used as it isor cut to sizes appropriate for the envisaged application.

The present invention has led to the production of bicomposite materialsin which a layer of fibrous polymer, notably non-denatured collagen,which is extremely porous and may be very thick, to form an efficientcompress or sponge, is very closely bound to a thin collagenic film,which is well delimited and has suitable properties and dimensions.

It was then established that such a two-layer material displayed a setof particularly surprising hemostatic, anti-post-operative adhesion andbiodegradability qualities.

The biomaterial obtained is easy to handle. It does not stick tosurgical instruments or gloves when dry.

It displays acceptable mechanical resistance while retaining a certainflexibility, provided by the hydrophilic elements in the film ofcomposite material.

The material according to the invention is a local hemostatic, theactive principle of which is the polymer constituent, notablynon-denatured collagen or oxidized cellulose, which contributes, likeendogenous collagen, to the hemostatic and healing process. It ispreferably applied with pressure to the site of haemorrhage untilhemostasis is obtained. The blood is absorbed by the porous layer ofmaterial and concentrated under the material with the film of materialacting as a seal barrier. On contact with the polymer, it is transformedinto a hemostatic plug and/or a clot.

The material very quickly adheres to the bleeding wound, through theformation of a hemostatic plug and/or clot by the polymer.

It is thought that the considerably improved hemostatic properties ofthe compress according to the invention are notably due to thepossibility of absorbing a very large quantity of blood while preventingit from spreading either transversally or in the plane of thebiomaterial. In addition, the diffusion of blood through the porouscompress, within the area marked by the wound, increases the area ofcontact between the hemostatic substance and the platelets. It thusaccelerates hemostasis by playing on the various ways of obtainingcoagulation, the final phase of which leads to the formation of anetwork of platelets and fibrin reinforcing the compress's adhesion tothe wound.

On the contrary, the two-layer collagenic materials of the prior artdescribed above, are insufficiently porous so that the blood cannotpenetrate. This favours the lateral leakage of blood under the compresswhich does not provide good adhesion. Because of this, it is much harderto stop the bleeding.

The bicomposite collagenic material according to the invention isparticularly suitable for preventing post-operative adhesion,particularly in bleeding wounds, because the film prevents adherence,the composite material providing good adhesion in such wounds and thereis no blood at the interface.

Apart from their hemostatic properties and the prevention ofpost-operative adhesions, the collagenic material of the presentinvention facilitates healing because of its composite structure,combining a highly porous polymer layer and a collagenic film.

The porous part of the material can easily be colonized by thesurrounding cells. The film protects the healing wound for several daysas it forms a barrier to bacteria and micro-organisms.

The power of the film of the material to prevent adhesion is alsoreinforced by the polymer used for the porous layer of materialaccelerating healing of the wound.

According to the invention, the bicomposite collagenic material istherefore useful for hemostasis and the prevention of post-operativeadhesions on bleeding wounds, while facilitating healing.

In addition, the macromolecular hydrophilic additive is eliminated bydiffusion through the collagenic material, in a few days, the swellingof this material promoting degradation of the collagenic film in lessthan a month.

The bicomposite material according to the invention can also be used topromote healing. Its very open porous structure promotes rapid cellularcolonization. The film isolates the porous part to make it accessible tospecific cells.

As an example, fibroblasts can be cultured in the porous part of thematerial, in vitro, and epithelial cells can be cultured on the filmmaking two temporarily separate compartments.

However, although this is not preferred, a film of collagenicconstituent and a non-compacted porous compress can be bound by abiocompatible, biodegradable and non toxic adhesive agent, so long asthis agent can provide a sufficiently strong bond between the film andthe compress, although it is only present in small quantities.

Examples of adhesive agents are surgical glues, notably fibrin andcollagenic glues described in the patent Tardy et al. U.S. Pat. No.5,618,551 and application WO 98/15299.

This invention will now be described in detail with the aid ofnon-limiting examples showing different possible combinations of thematerials and their hemostatic powers and ability to preventpost-operative tissular adhesions.

According to the invention, a compress can be made which is then cut tothe size and shape required, or a biomaterial prepared which has thesize and shape of the patch required.

EXAMPLES Example 1

Preparation of Collagen Compresses With a Neutral pH:

The collagen used in type I bovine collagen, extracted from calf dermis,and possibly rendered soluble through pepsin digestion and, purified bysaline precipitation, using the techniques already described. Type I ortype III human collagen or a mixture of these in any proportions can beused in the same way.

A 10 g/l solution of collagen is prepared by dissolving 23 g of dampcollagen (12% humidity) in 2070 g of ultrafiltered water, at an ambienttemperature below 25° C. It is neutralized using sodium hydroxide to aneutral pH, which leads to precipitation of the collagen.

The suspension is then poured onto freeze-dry plates, with 0.5 to 1g/cm² and dehydrated by freeze-drying, using one cycle lasting about 24hours.

Finally, in a variant, the freeze-dried collagen compress can be heatedto 60° C. for several hours (4 to 15) which provides it with bettercohesion and mechanical resistance in certain applications.

Example 2

Preparation of Collagen Compresses With a pH of 5-5.5:

The preparation of collagen compresses with a pH 5-5.5 helps to limitthe collagen precipitation phenomenon. It is prepared as in example 1,the only difference being the neutralization of the collagen solutionwith sodium hydroxide at a pH close to collagen's isoelectric point,i.e. 5 and 5.5.

Example 3

Preparation of Collagen Compresses With an Acid pH:

Slightly acid compresses are prepared as in example 1, the onlydifference being that the collagen solution is not neutralized, whichavoids any collagen precipitation.

Example 4

Preparation of a Solution of Oxidized Collagen:

The 30 g/l oxidized collagen used for this example, is preparedaccording to patent FR-A-2 715 309. Type I bovine collagen is used,extracted from calf dermis by solubilization at an acid pH, or pepsindigestion, and purified by saline precipitation according to thetechniques already described.

The products marketed by COLLAGEN Corp. under the names VITROGEN® orZYDERM®, may be used in this application.

Dry collagen fibres are used for preference, obtained by precipitationof an acid solution of collagen by adding NaCl, then washing and dryingthe precipitate obtained using aqueous solutions of acetone inconcentrations increasing from 80% to 100%.

Type I or type III human collagen or any mixture of these can be used inthe same way.

The 30 g/l solution of collagen is prepared by dissolving it in 0.01 NHCl. Its volume is 49 liters. Periodic acid is added to it at a finalconcentration of 8 mM, i.e. 1.83 g/l. Oxidation takes place at anambient temperature close to 22° C. for 3 hours away from light.

Then an equal volume of a solution of sodium chloride is added to thesolution to obtain a final concentration of 41 g/l NaCl.

After waiting for 30 minutes, the precipitate is collected bydecantation through a fabric filter, with a porosity close to 100microns, then washed 4 times with a 41 g/l solution of NaCl in 0.01 NHCl. This produces 19 kg of acid saline precipitate. This washingprocess eliminates all traces of periodic acid or iodine derivativesduring oxidation of the collagen.

Then, several washes in an aqueous solution of 80% acetone are used toconcentrate the collagen precipitate and eliminate the salts present.

A final wash in 100% acetone is used to prepare 3.6 kg of a very denseacetone precipitate of acid, oxidized, non-reticulated collagen, with notrace of undesirable chemical products.

The acetone paste is diluted with apyrogenic distilled water at 40° C.,to obtain a 3% concentration of collagen, for a volume of 44 liters.This suspension of oxidized collagen is used to prepare porouscompresses in a similar way to examples 1,2 and 3.

Example 5

Preparation of a Solution of Heated Collagen:

A collagen gel of neutral pH and concentration close to 50 g/l is heatedto 45° C. for 10 minutes to fluidify it.

4 volumes of air or other gas are incorporated into the solution ofheated collagen through 2 syringes mounted opposite each other andconnected to produce the emulsion, by successively pulling and pushingthe plungers, which mix the respected contents of each syringe evenly.

The emulsion is prepared on freeze-dry plates and gelled by cooling,then frozen and freeze-dried.

Example 6

Preparation of a Solution of Oxidized, Heated Collagen Designed to Forma Film

The suspension of a volume of 44 liters described in example 4, isheated for 30 minutes at 50° C., then filtered under sterile conditionsthrough a membrane of 0.45 micron porosity in a drying oven at 40° C.

As soon as this solution is homogeneous and at 35° C., a sterileconcentrated solution of PEG 4000 (polyethylene glycol with a molecularweight of 4000 Daltons) and glycerine is added to it to produce a finalconcentration of 0.9% PEG, 0.54% glycerine and 2.7% oxidized collagen.

As soon as these additions have been made, the pH of the solution isadjusted to 7.0 by adding a concentrated solution of sodium hydroxide.

Example 7

Preparation of a Solution Including a Mixture of Non-oxidized, HeatedCollagen and Oxidized Collagen, Designed to Form a Film:

A variant of the preparation of collagen solution used for the film, isto take heated non-oxidized collagen or a mixture of heated oxidizedcollagen, prepared as in example 6, and heated non-oxidized collagen, inany proportions.

The collagen used for preparing non-oxidized, heated collagen is type Ibovine collagen, extracted from calf dermis, possibly solubilized bypepsin digestion and purified by saline precipitation using thetechniques already described. Type I or type III human collagens ormixtures of these in any proportions can be used in the same way.

A 30 g/l solution of non-oxidized, heated collagen is prepared bydissolving 65.2 g of damp collagen (12% humidity) in 1940 g ofultrafiltered water at 42° C. A sterile concentrated solution of PEG4000 (polyethylene glycol with a molecular weight of 4000 Daltons),glycerine and possibly oxidized, heated collagen prepared as in example6 is added to this solution at 42° C. to produce a final concentrationof 0.9% PEG, 0.54% glycerine and 2.7% total collagen. The pH of thesolution is adjusted to 7.0, by adding a concentrated solution of sodiumhydroxide.

Example 8

Preparation of an Acid Solution of Non-oxidized Heated Collagen Designedto Form a Film:

An acid solution of heated, non-oxidized collagen, for the film, isprepared as in example 7, with the following differences:

-   -   i) the collagen used is only non-oxidized heated collagen, the        preparation of which is described in example 1;    -   ii) the mixture used for the film, of which the final        concentrations of PEG, glycerine and collagen are 0.9%, 0.54%        and 2.7% respectively, is acid.

Example 9

Preparation of a Bicomposite Material from a Collagen Compress:

The collagen solution destined to form the film, as described inexamples 4 to 7, is poured in a thin layer with a density of 0.133 g/cm²on a flat hydrophobic support such as PVC or polystyrene, at an ambienttemperature close to 22° C.

A collagen compress, prepared as in examples 1, 2 or 3 is applieduniformly to the solution of heated collagen, 5 to 20 minutes after itwas poured onto the support. This waiting time is the collagen solutiongelling time, required for application of the collagen compress, toprevent it dissolving or becoming partially hydrated in the liquidcollagen.

Penetration of the compress into the gelled collagen solution is judgedto be less than 0.5 mm.

The material is then dehydrated in a jet of sterile air, at ambienttemperature, which leads to evaporation in about 18 hours.

The bicomposite material obtained is easy to remove from the support.

It can be cut to the dimensions required for the application concerned,without weakening it.

The bicomposite material is then put into an airtight doublepolyethylene bag.

The unit is sterilized by gamma irradiation or electron beam (beta)irradiation at a dose of between 25 and 35 KGy.

The material is stable at ambient temperature.

The presence of glycerine in the material essentially helps to make thefilm more flexible and facilitates its use. The material can be preparedwithout glycerine.

The use of PEG 4000 as macromolecular hydrophilic agent is not limiting.PEG 3000, PEG 6000 or polysaccharides such as soluble starch (OSI,France) and Dextran T40 (Pharmacia Fine Chemicals, Sweden) can be usedinstead.

FIGS. 1 and 2 are photographs taken under scanning electron microscope,enlarged by 40 and 200 times respectively, illustrating the structure ofthe bicomposite material prepared as indicated above.

FIG. 1 shows a specimen of example 9 made from the compress as inexample 1 prepared from pepsinated collagen, the film being produced asin example 6.

FIG. 2 shows a specimen of example 9 made from the compress as inexample 3, the film being produced as in example 8.

Example 10

Preparation of a Bicomposite Material Using an Oxidized CelluloseCompress:

The procedure is the same as for example 9 but using a porous compressbased on oxidized cellulose as is available on the market under the nameInterceed® or Surgicel®.

1. A method for obtaining a bicomposite material which has two closelybound layers and is biocompatible, non-toxic and biodegradable in lessthan one month, said method comprising the steps of: (i) pouring asolution of collagen or gelatin onto an inert support so as to form a 30μm to less than 100 μm-thick layer film after drying step (iii); (ii)applying to the solution during gelling of the collagen or gelatin apolymeric porous fibrous layer having a density of no more than 75mg/cm², a pore size from 20 μm to 300 μm and a thickness of 0.2 cm to1.5 cm; and (iii) drying or leaving to dry the material obtained fromstep (ii) to provide said bicomposite material.
 2. The method accordingto claim 1, wherein the solution of collagen in step (i) has aconcentration of collagen of between 5 and 50 g/l.
 3. The methodaccording to claim 2, wherein the solution of collagen in step (i) is anacid solution of native collagen.
 4. The method according to claim 1,wherein the solution of collagen in step (i) includes collagen modifiedby oxidative cleavage.
 5. The method according to claim 4, wherein thesolution of collagen in step (i) is modified by treatment with periodicacid or one of its salts.
 6. The method according to claim 1, whereinthe solution of collagen in step (i) is heated to a temperature ofbetween 40° and 50° C.
 7. The method according to claim 1, wherein atleast one macromolecular hydrophilic additive; chemically unreactivewith respect to the collagen or gelatin, is added to the solution ofcollagen in step (i).
 8. The method according to claim 7, wherein theconcentration of hydrophilic additive(s) is 2 to 10 times less than theconcentration of collagen in the solution in step (i).
 9. The methodaccording to claim 7, wherein glycerine is added to the solution ofcollagen in step (i).
 10. The method according to claim 9, wherein theconcentration of glycerine is between 3 and 8 g/l and does not exceedone third of the concentration of collagen of the solution in step (i).11. The method according to claim 1, wherein the collagen solution instep (i) is an aqueous solution containing 2 to 10% of collagen orgelatin, 0.6 to 4% of hydrophilic additive(s) and 0.3 to 2.5% ofglycerine.
 12. The method according to claim 1, wherein the solution instep (i) is neutralized.
 13. The method according to claim 1, whereinthe support in step (i) is a PVC or polystyrene support.
 14. The methodaccording to claim 1, wherein the solution in step (i) has a density ofbetween 0.1 and 0.3 g/cm².
 15. The method according to claim 1, whereinthe collagen or gelatin solution in step (i) is poured at a temperatureof 4 to 30° C.
 16. The method according to claim 1, wherein thepolymeric porous fibrous layer in step (ii) is made of collagen.
 17. Themethod according to claim 16, wherein the polymeric porous fibrous layerof step (ii) is prepared from an aqueous acid solution of collagen, theconcentration of which is 2 to 50 g/l when the collagen is notdenatured.
 18. The method according to claim 17, wherein the aqueousacid solution of collagen is neutralized to a pH of around 7 to
 8. 19.The method according to claim 17, wherein the solution of collagen usedto prepare the polymeric porous fibrous layer of step (ii) isfreeze-dried.
 20. The method according to claim 19, wherein the solutionof collagen used to prepare the polymeric porous fibrous layer of step(ii) is spread in a layer with a density of between 0.2 and 1.5 mg/cm²for freeze-drying.
 21. The method according to claim 1, wherein thepolymeric porous fibrous layer of step (ii) is made of polysaccharide.22. The method according to claim 1, wherein the polymeric porousfibrous layer of step (ii) is made of polysaccharide modified byoxidation of the alcohol functions into carboxylic function.
 23. Themethod according to claim 1, wherein, when the polymeric porous fibrouslayer is applied to the solution of collagen or gelatin during gelling,the polymeric porous fibrous layer of step (ii) is allowed to penetratefor around 0.05 to 2 mm in the gel which is forming.
 24. The methodaccording to claim 1, wherein the material obtained is dried in a jet ofsterile air in step (iii).
 25. The method according to claim 1, whereinthe polymeric porous fibrous layer is produced by freeze-drying acollagenic emulsion and a gas.
 26. The method according to claim 1,wherein the material obtained is sterilized in step (iii).
 27. Themethod according to claim 7, wherein the macromolecular hydrophilicadditive has a molecular weight of between 3,000 and 20,000 Daltons. 28.The method according to claim 7, wherein the macromolecular hydrophilicadditive is polyethylene glycol.
 29. The method according to claim 7,wherein the hydrophilic additive is chosen from the group consisting ofpolysaccharides and mucopolysaccharides.
 30. The method according toclaim 7, wherein the hydrophilic additive is an oxidized polysaccharide.